The present invention concerns a method for the removal and inactivation of viruses from biological preparations.
Biologically derived liquid preparations such as blood and plasma preparations are used as raw materials from which a plurality of biologically useful compounds can be purified. Examples of such compounds include immunoglobulin, factor VIII, albumin, xcex1 1 anti trypsine, Factor IX, factor XI, PPSB, fibrinogen, and thrombin (prothrombin). In addition, various biological products such as hormones, growth factors, enzymes and ligands are isolated from biological preparations obtained from cell cultures.
The cells used in the production of these useful materials may be either wild-type cells from various animal sources, or alternatively, genetically engineered prokaryotic or eukaryotic cells. Where the biological materials obtained from these liquid preparations are to be administered to humans for therapeutic purposes, in particular by intravenous administration, the sterility of the preparation is of major concern. Thus, great efforts are invested in the inactivation of viruses, such as hepatitis viruses and HIV viruses, which may be present in these preparations.
Lipid coated viruses are effectively inactivated by treatment with non-ionic biocompatible solvents and detergents. Methods for virus inactivation by solvent-detergent applications are described, for example, in EP 0131740. However, non-lipid coated viruses cannot be inactivated by solvent-detergent treatments, thus, other inactivation methodologies have to be used for their inactivation. These include the application of heat (pasteurization), the application of irradiation such as short ultra violet light (UVC) or gamma radiation, as well as eliminating by physical means, e.g., the filtration of the preparation through very narrow filter holes so as to remove viruses by size exclusion (nanofiltration).
Noreen et al., (Biologicals, 26:321-329 1998) examines the use of the Memberg Microporous membrane hollow fibers (Planova) 35 mn filters to reduce potential loads of both non-enveloped and enveloped viruses, prior to the solvent detergent treatment in a 7% IVIg solution. In the above study, nanofiltration was validated for the removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhea virus (BVDV), Feline calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The study showed a complete reduction (to the limit of detection assay) of all viruses larger than 35 nm. Interestingly, even smaller viruses such as EMC and HAV were at least partially removed by this method of filtration.
These studies led to the use of nanofiltration for the removal of viruses from biological preparations. However, in cases where it was desired to combine both viral inactivation by the solvent-detergent method, (in order to inactivate lipid-coated viruses) as well as nanofiltration elimination (for the size exclusion and hence removal of non-lipid coated viruses), it was discovered that the nanofiltration step had to proceed the solvent-detergent application, particularly, where the solvent-detergent was to be removed by oil extraction and C-18 reverse phase resin. The reason for this was that after extraction of the solvent-detergent, there are always traces of small oil droplets, as well as residues of solvent-detergent, which bind to the hydrophilic part of the resin. Moreover, some of the proteins purified from the biological preparation may be modified during the purification process, and the altered proteins can form dimers and polymers which change in the hydrophobicity of the altered protein. These traces of oil droplets, protein dimer aggregates and the mixture of oil and protein residues tend to block the small holes of the nanofilter, thus considerably increasing the time of the filtration, requiring the frequent replacement of expensive filters, and generally decreasing the yield of the product.
The residues of oil droplets (contaminants), which tend to block nanofilters, were removed by using either a chromatography mechanism of molecular exclusion or by hydrophobic chromatography. However, none of the conventional methods for the removal of solvent detergent has yet addressed the issue of dimerization or aggregation caused by the solvent detergent, and this problem still remains. Removal of the solvent-detergents from biological liquid preparations, such as immunoglobulin preparations, is generally carried out by using gel chromatography. U.S. Pat. No. 5,094,960 and U.S. Pat. No. 5,648,472 concern the removal of solvent-detergent from immunoglobulin preparations without using gel reverse phase (hydrophobic) chromatography.
Two other patents have been granted to processes which indicate that the removal of solvent detergent affects the stability of a liquid IVIg product (U.S. Pat. No. 5,094,960, U.S. Pat. No. 4,789,545 and U.S. Pat. No. 5,648,472). G. Werner and P. Selosse (U.S. Pat. No. 5,648,472) describe a process for preparing envelope virus-inactivated immunoglobulin solutions suitable for intravenous application, comprising treating the immunoglobulin with TnBP and/or Tritonx100(trademark) (octylphenol ethylene oxide condensate; CAS 9002-93-1), followed by an extraction using biologically compatible vegetable oil, when TnBP and/or Tritonx100(trademark) and vegetable oil are subsequently removed by solid-phase extraction on hydrophobic materials. This indicated that the combination of vegetable oil extraction of IVIg followed by chromatography through a hydrophobic column produces a very stable liquid solution of immunoglobulins, even at elevated temperature. This patent claimed that IVIg preparation is prepared in accordance to two previous patents:
Bonomo, 1992, (U.S. Pat. No. 5094960) teaches the use of solid phase extraction and Bulk C-18 packing from Waters, Inc. as a preferred resin. Woods 1986 (U.S. Pat. No. 4,789,545), features the removal of solvent detergent by naturally occurring oils, however the products obtained by this method resulted in the unstable preparation of intravenous immunoglobulins.
Guerrier et al (Journal of Chromatography B 664:119-125 (1995)) describes a specific sorbent which is intended to remove solvent-detergent mixtures from virus-inactivated biological fluids. The solvent-detergent removal (SDR) HyperD(trademark) sorbents were supplied by Biosepra (Ceroy-Saint-Christophe, France). This chromatographic packing was made of silica beads in which the pore volume was filled with a three-dimensional cross-linked hydrophobic acrylic polymer.
The present invention is based on the finding that a method for the inactivation of viruses present in biological preparations comprising first treating the preparation by solvent-detergent, after which the solvent-detergent is extracted by the use of vegetable oil, in a combination with/without solid phase extraction (specifically using bulk C18 as recommended by U.S. Pat. No. 5,648,472) resulted in an immunoglobulin liquid preparation which was very difficult to pass through the Planova (trademark)Filter 35 nm. Thus, it was realized that such a procedure was unsuitable for the efficient inactivation of viruses.
In accordance with the present invention, it was surprisingly discovered that the combination of solvent-detergent treatment, followed by chromatography utilizing SDR HyperD(trademark) (ion-exchange resin) from Biosepra, facilitated the passage of the IVIg at a high flow rate through Planova Filters 35 nm, and resulted in a liquid preparation devoid of active viruses, while featuring a very high yield. In accordance with the present invention, it was further found that the problem of dimerization of immunoglobulins can be partially solved by reducing the pH to 4.0 before the nanofiltration step.
Thus, the present invention concerns a method for the inactivation of viruses present in a liquid preparation, comprising the steps of:
(i) contacting the biological liquid preparation with a solvent-detergent combination at concentrations and under conditions which are sufficient to inactivate lipid-coated viruses;
(ii) removing solvent-detergent traces from the liquid preparation by passing the liquid preparation obtained in (a), on a chromatographic packing composed of silica beads whose pore volume is filled with three-dimensional cross-linked hydrophobic acrylic polymer; and
(iii) passing the liquid product of step (b) through a filter having a pore size ranging from about 15 nm to approximately 70 nm.
Optionally, after step (a) comes a step of extraction of the solvent-detergent by a hydrophobic composition such as oil, for example, a biologically compatible vegetable oil. The removal of the chromatographic packing in step (b) is thus also useful in the elimination of the oil traces and oil related impurities.
The term xe2x80x9cinactivating virusesxe2x80x9d refers both to the situation wherein viruses are maintained in the solution but are rendered non-viable (for example, by dissolving their lipid coat), as well as to the physical removal of the viruses from the liquid preparation (for example, by size exclusion). Thus, in the context of the invention, this term refers both to viral destruction and viral removal.
The term xe2x80x9cbiological liquid preparationxe2x80x9d refers to any type of liquid preparation obtained from a biological source. This typically includes preparations obtained from body fluids such as blood, plasma and urine, as well as liquids obtained from cell cultures, containing biological substances secreted by the cells into the preparation, or containing substances which originally were present inside the cells, and were released to the liquid preparation due to various manipulations such as the lysing of the cells.
The method of virus inactivation, in accordance with the invention, may be used for a plurality of utilizations such as: the isolation of various proteins including immunoglobulins, factor VIII, albumin, xcex1 1 anti trypsine, Factor IX, factor XI, PPSB, fibrinogen, and thrombin (prothrombin) and others; the isolation of genetically engineered proteins from cell cultures, the isolation of hormones, growth factors, enzymes, clotting factors, receptors, and other biologically active copolymers and the like.
In accordance with a preferred embodiment of the invention, the biological liquid preparation is intended for the isolation of immunoglobulins which are to be purified therefrom and is obtained by resuspending Paste II, from plasma fractionation, in water, adjusting the pH of the preparation and ultrafiltering and diafiltering the resulting products by using membrane filters to give a desired protein concentration.
The solvent-detergent combination used to deactivate lipid coated viruses may be any solvent-detergent combination known in the art such as TnBP and Triton X-100(trademark), Tween 80(trademark) (CAS 9005-65-6; polyoxyethylene sorbitan monooleate) and Sodium cholate and others. The concentration of the solvent detergents should be those commonly used in the art, for example,  greater than 0.1% TnBP and  greater than 0.1% Triton X-100(trademark). Typically, the conditions under which the solvent-detergent inactivates the viruses consist of 10-100 mg/ml of solvent detergent at a pH level ranging from 5-8, and a temperature ranging from 2-37xc2x0 C. for 30 min. to 24 hours. However, other solvent detergent combinations and suitable conditions will be apparent to any person versed in the art.
After undergoing solvent-detergent treatment, the bulk of the solvent-detergent is removed by the use of an SDR resin (solvent-detergent removal), which is a chromatographic packing made of silica beads in which the pore volume is filled with a three-dimensional cross-linked hydrophobic acrylic polymer. An example of such an SDR is the HyperD(trademark) resin supplied by Biosepra.
The resulting preparation is then passed through a nanofilter, having a pore size of less than 70 nm, preferably between 15 and 50 nm. The precise size of the pore should be determined in accordance with the protein which has to be maintained in the liquid preparation, and the size of the viruses which have to be eliminated by size exclusion.
The method of the present invention has the following advantages over other methods of inactivation of viruses in which the nanofiltration step precedes the treatment with a solvent detergent:
(1) The amount of viruses which adhere to the pores of the filter is smaller (since some of the viruses were eliminated by the solvent-detergent treatment) so that the replacement of filters, which is an expensive part of the operation, is decreased.
(2) The time course of the procedure is decreased, since the liquid preparation, after being treated with the solvent-detergent and the SDR extraction, passes much more quickly through the nanofilter than had this nanofiltration step had been carried out as the first step of the method.
(3) Blocking the filters during the production resulted usually in a significant decrease in yield. The yield of the present invention is about 97-100%.
(4) The hydrophobic filling of the SDR resin decreases the dimer""s concentration in the immunoglobulin solution. Once the immunoglobulin is depleted of its dimers, the low pH levels reduce the rate of new dimer formation by charging the molecules with additional negative charge which repels then from each other.
The dimerization problem where the preparation is an immunoglobulin preparation is solved partially by reducing the pH level to a pH below 5.